The toxins of bacterial cell walls (Henkel et al, EXS. (2010) 100: 1-29) have been associated with health related issues, such as septic shock, fever and malaise (V. Liebers et al., Am J Ind Med. (2006) 49(6): 474-91). Examples of Gram-negative cell wall toxins associated with health concerns are endotoxins, such as lipopolysaccharide (LPS), peptidogylcans, and fimbriae; and Gram-positive cell wall toxins associated with health concerns are lipoteichoic acid (LTA) and peptidoglycans. There are many other bacterial toxins, such as enterotoxins and exotoxins, as reported in Henkel et al, EXS. 2010; 100: 1-29. For the oral environment, the LPS and LTA seem to be the dominant drivers of a bacterial induced immune response, or at least the best characterized. The immune response mounted by the body in response to these toxins depends on the origin of the toxin and the exposure history of the individual to said toxin. The LPS is a component of Gram-negative bacteria that is different from strain to strain, as has been illustrated with the differences in virulence of E. coli (Raetz and Whitfield Annu. Rev. Biochem (2002) 71:635-700). LPS is composed of a lipid A fraction, core region, and may have an O-antigen. The Lipid A fraction's fatty acid composition has been shown to determine its virulence in response to its interaction with the Toll-like 4 (TLR4) receptor. The LTA has been linked to various inflammatory responses (Y. Yokoyama, et al., Acta Otolaryngol Suppl. (1996) 523:108-111) and associated with Toll-like receptor 2 (TLR2) activation. It is widely believed that only lysed bacteria liberate LPS that can initiate an inflammatory response (CA2323630). However, Zhang et al. showed that growing bacteria secrete LPS at a level in proportion to their growth phase (H. Zhang et al. (1998) Infection & Immunity, 66(11), 5196-5201). Therefore, even a small fraction of the plaque left on the teeth after brushing could seed the inflammatory cascade due to the release of LPS from the gram negative bacteria present in the plaque.
Methods of detecting specific microbial species have been demonstrated in the art. In US Pub. No. 2012/019735A1, methods were proposed to distinguish disease-causing bacteria via spectrophotometric methods. Though they were able to show the presence of specific microbes, their invention would not allow the user to determine the virulence level of a specific site. Further, their method requires the microbes to be cultured in the lab to obtain a sufficient quantity of LTA or LPS for detection. Thus, their invention lacks the ability to detect the non-culturable species present, and would not allow for measuring the toxicity of biological samples.
In U.S. Pat. No. 5,175,089, the use of the Limulus amebocyteH lysate (LAL) endotoxin (LPS) assay was applied to the determination of the amount of endotoxin in the periodontal pocket. Though they were able to show overall amounts of endotoxin present, they lacked the ability to differentiate diseased versus healthy endotoxin and they were unable to quantify the level of virulence of the endotoxin. Further, their invention limited them to the Gram-negative endotoxin, as the LTA is not detectable via the LAL kit.
TLR signal transduction involves association of the chaperonin 10 with a Toll-like receptor in an activation cluster. In US Pub. No. 2009/0047240, the chaperonin 10 (Cpn10) antibodies were used to modulate the clustering of Cpn10 in a cell line, murine RAW264, thus changing the signal transduction of TLR. Though they showed TLR-4, 7, and 9 reporter genes in an HEK cell line, their system would not allow for a more sensitive or low level detection needed for microbial populations with weaker activating LPS, since those genes were under the control of the NFkB binding sites only (a minimal promoter). Their system lacks the sensitivity needed to differentiate biological systems with multiple microbial species and no dominant organism present. Further, their system needs strong NFkB activators to overcome the weak promoter used in their system, thus unable to pick up weaker TLR LPS agonists, such as LPS from Porphyromonas gingivalis. Additionally, their system lacked the ability to detect TLR3 agonists, which would be deleterious to the characterization of an inflammatory disease, such as gingivitis.
US Pub. No. 2007/0160544 describes a method for determining orally deleterious bacteria. Their method calls for contacting a gingival cell with bacteria or a bacterial component and measuring an inflammatory marker. According to US Pub. No. 2007/0160544, the presence of a marker indicates inflammation and the labeling of a bacterium as deleterious. Conversely, they say that the absence of a marker indicates the bacterium is not a problem. Though they cited Toll-like receptors, which were known in the art as part of the pathway to generate cytokines, their method would have only allowed for determining the presence of a cytokine. The methods outlined in '544 were predicated on the workings of Dongari-Bagtzoglou and Ebersole in J. Perio. Res. 31: 90-8 1996, where Dongari showed that human gingival fibroblast cells could be used to screen bacteria for an inflammatory response by measuring the production of either IL-1 beta, IL-6, or IL-8 cytokines. The '544 application was analogous to the work of Madiano et al., in J. Clinical Perio. 32: 57-71 2005, where Madiano reviewed the literature around bacterial virulence factors and disclosed the concept of gingival cells as methods to determine the anti-inflammatory properties of multiple bacterial components from several species of bacteria. Their system proposes the use of indicator materials (cytokines) generated from the host immune response by the activation of any Toll-like receptor. Though their proposed system may give rise to a cytokine, as was demonstrated in the literature (Shapira et al., J. Dent. Res. 76: 1381-6 1997), their proposed invention would not allow for quantification of the specified cytokine, nor would it allow for determination of the virulence potential of the bacterial components. Thus, what is needed is an invention that can quantify virulence and determine level of potency.
Since oral cells contain one or more of the receptors to which a bacterial virulence factor would activate, screening on the individual receptors requires the use of engineered cells, such as reporter cells containing the receptor gene of interest. What further complicates the use of native oral cells, such as gingival cells, is that the expression and activation of a receptor, such as a Toll-like receptor, is specific to the function of the cell. Gingival cells are less likely to respond to bacterial virulence factors, due to their constant contact with microbes in the dental plaque. Thus the need exists to have engineered cells where a direct response can be measured via a reporter system.
In addition to quantifying the virulence of microbial components and byproducts, there also exists a need for an in vitro screen of the inflammatory potential of organic and inorganic molecules, which would allow for pharmokinetic parameters to be determined.